1. Field of the Invention
The present invention relates to a method for analyzing a structure of glycoprotein or glycopeptide using a mass spectrometer capable of executing a second or higher order MS analysis.
2. Disclosure of the Related Art
For analyzing a structure of a glycoprotein or a glycopeptide, it is necessary to acquire information about (i) peptide sequence, (ii) sugar chain sequence, and (iii) sugar chain binding site.
In a method for analyzing a glycoprotein or a glycopeptide, sugar chains are separated from the glycoprotein or the glycopeptide (hereinafter, simply referred to as “glycoprotein”) in an enzymatic or chemical manner and then the resultant protein or peptide (hereinafter, simply referred to as “protein”) and sugar chains are separately analyzed. For such analysis, a mass spectrometer is used in these days. In mass spectrometry, a post-source decay (PSD) function and a collision induced dissociation (CID) function are utilized. These functions will give important structure information for sugar chains and fragments of a peptide.
As a method for separately analyzing proteins and sugar chains, for example, the following manner is exemplified. First, a glycoprotein obtained by two dimensional electrophoresis or a variety of chromatography is fragmented into peptides or glycopeptides with a protease, and further separated into sugar chains and peptides by glycanase digestion or β-dissociation. Then the resultant peptides are chemically tagged at an amino acid residue to mark the binding site of sugar chain, and then sequencing analysis of peptides and identification of sugar chain binding site are conducted by a peptide mass finger printing (PMF) based on MS analysis or by an MS/MS analysis based on the CID and PSD functions with a mass spectrometer. On the other hand, as to the sugar chains, a sequencing analysis of sugar chains is conducted by MS and MS/MS analyses using a mass spectrometer, by a multi-dimensional mapping using a HPLC, or by a sequential digestion with glycosidase.
Also an attempt is made to directly analyze a structure of glycoprotein in its native form without separating sugar chains as above described. For achieving this, an MSn analysis by a mass spectrometer is utilized. A Fourier transformation ion cyclotron resonance mass spectrometer (FTICR-MS) is known as a mass spectrometer that enables mass spectrometric analysis of glycoprotein. In such a mass spectrometer, an electron capture dissociation (ECD) function and an infrared multiphoton dissociation (IRMPD) function that were recently developed are specifically used. The ECD function gives information about sugar chain binding site and molecular weight of sugar chain because it can cut only a peptide backbone without destroying sugar chains. The IRMPD function gives sequence information of sugar chain because it can cut only sugar chains without cutting a peptide backbone. Therefore, a peptide sequencing analysis, a sugar chain sequencing analysis, and an analysis of sugar chain binding site with a FTICR-MS is conducted by combining these two functions.